Nontypable Haemophilus influenzae (NTHi) is a major pathogen in otitis media. It appears to be especially prominent in children with recurrent episodes of acute otitis media (AOM). The broad, long-term objective of this proposal is to define the various roles of lipopolysaccharide (LPS) as a virulence factor in pathogenesis of AOM, with the ultimate goal of developing an LPS inner-core glycoconjugate vaccine against NTHi. Prior efforts in vaccine development have been limited by diversifying selective pressures on surface exposed epitopes of NTHi; however, the use of conserved LPS inner-core may circumvent this limitation. The first specific aim is to identify phenotypic and genotypic changes in NTHi LPS expression during transition from asymptomatic carriage to middle ear disease, in order to identify LPS glycoforms that may have potential as additional vaccine target candidates. Animal modeling in conjunction with mass spectrometry techniques and microarray technology should enable the identification of such critical LPS glycoforms. Secondly, interaction between newly resolved LPS glycoforms and host defense mechanisms will be assessed, by flow cytometry and ELISA techniques. The final aim is to determine whether truncated inner-core LPS oligosaccharide structures, when conjugated to a protein carrier, are immunogenic and protective against experimental OM due to both homologous and heterologous isolates of NTHi in a chinchilla animal model. This collaboration between Boston Univ. Medical School, Institute for Biological Sciences NRC-Canada and Oxford University Institute for Molecular Medicine brings together synergistic expertise in microbial genetics and population biology, immunology, LPS structural chemistry, and animal modeling.